Precision fluorescently dyed polymeric microparticles and methods of making and using same

ABSTRACT

An improved method of making a series of bead or microsphere or particle populations characterized by subtle variation in a proportion or ratio of at least two fluorescent dyes distributed within a single bead of each population is provided. These beads, when excited by a single excitation light source are capable of giving off several fluorescent signals simultaneously. A set containing as many as 64 distinct populations of multicolored, fluorescent beads is provided and when combined with analytical reagents bound to the surface of such beads is extremely useful for multiplexed analysis of a plurality of analytes in a single sample. Thus, methods of staining polymeric particles, the particles themselves, and methods of using such particles are claimed.

This application is a continuation of application Ser. No. 10/401,549,filed Jul. 3, 2003 now U.S. Pat. No. 6,929,859, which is a continuationof application Ser. No. 09/172,174, filed Oct. 14, 1998, now U.S. Pat.No. 6,632,526, which claims the benefit of provisonal application60/061,938, filed Oct. 14, 1997 and 60,085,584, filed May 15, 1998.

1. FIELD OF THE INVENTION

The invention generally relates to multicolored, fluorescently stainedsmall particles of generally less than 100 μm in diameter. Disclosed aremethods of dyeing or staining such particles or microspheres with atleast two fluorescent dyes in such a manner that intra-sample variationof dye concentrations is substantially minimized. Specifically, theinvention relates to microspheres stained with at least two fluorescentdyes and methods of using said microspheres for a simultaneous analysisof a plurality of analytes.

2. BACKGROUND OF THE INVENTION

Fluorescent light emitting microparticles, microspheres, microbeads,beads, or particles are now quite common and are useful for a number ofpractical applications especially in combination with flow cytometrybased methods. As used hereinafter the terms: microparticles,microspheres, microbeads, beads, or particles are used interchangeablyand bear equivalent meanings. Often, these particles are labeled withjust one fluorescent dye. In general, such particles are made bycopolymerization process wherein monomers, e.g., unsaturated aldehyde oracrylate, are allowed to polymerize in the presence of a fluorescentdye, e.g., fluorescein isothiocynate (FITC), in the reaction mixture(see for example U.S. Pat. No. 4,267,234 issued to Rembaum; U.S. Pat.No. 4,267,235 Rembaum et al; U.S. Pat. No. 4,552,812, Margel et al.;U.S. Pat. No. 4,677,138, Margel).

One skilled in the art would recognize that two or more dyes of varyingproportions could be used to increase the permutation number of uniquecombinations of dyes in a single particle. These unique characteristics,i.e., emission wavelengths and fluorescence intensities could beextremely useful for multiparameter analysis of a plurality of analytesin the same sample. Three means of making multicolored, fluorescentbeads have been reported, including: (a) covalent attachment of dyesonto the surface of the particle, (b) internal incorporation of dyesduring particle polymerization, and (c) dyeing after the particle hasbeen already polymerized. All three methods have been disclosed in theprior art.

The examples of the first approach are in U.S. Pat. No. 5,194,300Cheung; U.S. Pat. No. 4,774,189 Schwartz which disclose fluorescentmicrospheres that are coated by covalently attaching either one or aplurality of fluorescent dyes to their surface. As such these methodsare unrelated to the instant invention dealing with incorporating dyesinto particles internally.

Second approach can be found in U.S. Pat. No. 5,073,498 to Schwartz,which discloses two or more fluorescent dyes added during polymerizationprocess and randomly dispersed within the body of the particle. However,when such particles are exposed to a single excitation wavelength onlyone fluorescent signal is observed at a time and thus these particlesare not useful for multiparameter analysis especially in a flowcytometry apparatus with a single excitation light source. The U.S. Pat.No. 4,717,655 issued to Fulwyler discloses two dyes mixed at fivedifferent ratios and copolymerized into a particle. Although fivepopulations of beads were claimed as being obtainable the fluorescentproperties of these beads were not provided, effectively preventing oneskilled in the art to make and use such beads. Thus, Fulwyler method isonly a conceptual method since it was not enabled. Furthermore, any ofthese two methods are unrelated to the instant invention dealing withincorporating fluorescent dyes into already polymerized particles.

The principle of the third method, i.e., internally embedding ordiffusing a dye after a particle has been already polymerized wasoriginally described by L. B. Bangs (Uniform Latex Particles; SeragenDiagnostics Inc. 1984, p. 40) and relates to the instant invention as itconsists of adding an oil-soluble or hydrophobic dye to stirredmicroparticles and after incubation washing off the dye. Themicrospheres used in this method are hydrophobic by nature. This allowsadopting the phenomenon of swelling of such particles in a hydrophobicsolvent, which may also contain hydrophobic fluorescent dyes. Onceswollen, such particles will absorb dyes present in the solvent mixturein a manner reminiscent to water absorption by a sponge. The level andextent of swelling is controlled by incubation time, the quantity ofcross-linking agent preventing particle from disintegration, and thenature and amount of solvent(s). By varying these parameters one maydiffuse a dye throughout particle or obtain fluorescent dye-containinglayers or spherical zones of desired size and shape. Removing thesolvent terminates the staining process. Microparticles stained in thismanner will not “bleed” the dye in aqueous solutions or in the presenceof water-based solvents or surfactants such as anionic, nonionic,cationic, amphoteric, and zwitterionic surfactants.

U.S. Pat. No. 5,723,218 to Haugland et al. discloses diffusely dyeingmicroparticles with one or more dipyrrometheneboron difluoride dyes byusing a process, which is essentially similar to the Bangs method.However, when beads internally stained with two separatedipyrrometheneboron dyes, were excited at 490 nm wavelength, theyexhibited overlapping emission spectra, meaning that beads weremonochromatic but not multicolored. U.S. Pat. No. 5,326,692 Brinkley etal; U.S. Pat. No. 5,716,855 Lerner et al; and U.S. Pat. No. 5,573,909Singer et al. disclose fluorescent staining of microparticles with twoor more fluorescent dyes. However, dyes used in their process hadoverlapping excitation and emission spectra allowing energy transferfrom the first excited dye to the next dye and through a series of dyesresulting in emission of light from the last dye in the series. Thisprocess was intended to create an extended Stokes shift, i.e., a largergap between excitation and emission spectra, but not the emission offluorescence from each dye simultaneously. Thus, due to various reasonssuch as dye-dye interaction, overlapping spectra, non-Gaussian emissionprofiles and energy transfer between neighboring dyes the demand formulticolored beads simultaneously emitting fluorescence at distinctpeaks was not satisfied. Zhang et al. (U.S. Pat. No. 5,786,219) devisedmicrospheres with two-color fluorescent “rings” or microspherescontaining a fluorescent spherical “disk” combined with a fluorescentring. Nevertheless, such beads, designed for calibration purposes,cannot be used in multiparameter analysis since two dyes were mixed onlyat one fixed ratio. As mentioned above in regard to U.S. Pat. No.4,717,655 issued to Fulwyler, the highest number of dyes ratios everattempted with at least two dyes never exceeded five. Thus, until thereduction to practice of the present invention there were no reliablemeans of creating a series of microsphere populations or subsets inwhich at least two dyes were mixed at variable, precisely controlledratios and were proven, upon exposure to a single excitation wavelength,to emit multiple fluorescent signals of variable intensity and atspaced, optically distant wavelengths.

In other words, the prior art failed to provide a reproducible methodthat would allow one skilled in the art to make a plurality of definedsubsets of stained multicolored microparticles distinguishable by asubtle variation in fluorescence signal resulting from the combinationof various dyes of distinct color and having variable intensity of coloremission. As used hereinafter the term stained microspheres means that aplurality of dyes, which are used to stain a microsphere, are eitheruniformly diffused throughout the body of said microsphere or penetratedsaid microsphere in a manner that results in formation of fluorescentrings, disks, and other geometrically distinct patterns.

Clearly, it would be an important improvement to the art to have a meansof precisely dyeing or staining a particle with two or more dyespremixed in a series of predetermined ratios and to have a collection ofsuch dyed microspheres for use in multiparameter applications. Thisprecision in dyeing process is commonly expressed as the coefficient ofvariation, which is the ratio of the standard deviation to the meanintensity of the fluorescent particle population. By minimizing thisvalue, more subsets or populations of non-overlapping, distinctly dyedparticles can be obtained. It would be a further advance in the art ifthe methods were repeatable or reproducible to within a minimalvariation, preferably no more than about a 20% intra-sample variation,more preferably no more than about a 15% variation, and most preferablyno more than about a 8% variation.

3. SUMMARY OF THE INVENTION

An improved method is described for incorporating two or morefluorescent dyes into already polymerized microspheres. The amount ofeach dye absorbed by the microsphere is precisely controlled so as togive rise to two or more reproducible fluorescent signals of preciseintensities and emission peaks within a given population of particles. Aseries of such populations or subsets of beads are dyed in batches eachone of them having predetermined ratio or proportion of two or morefluorescent dyes. Due to novel and improved method of staining, theparticle-to-particle variation in the same batch is greatly reduced,which allows producing an unprecedented number of distinct populationsof multicolored, fluorescent microspheres residing within opticallyuniform, tightly defined cluster.

Accordingly, a set containing optically distinct precision stainedmicrospheres is also claimed which would be useful for simultaneousanalysis of a plurality of analytes in a same sample. In other words,said beads will provide a lot more than the use of stained beads foundin the prior art since the number of analytes that can be measuredsimultaneously in a single tube, using a single sample aliquot isdrastically increased. The fluorescent microparticle obtained by theinventive staining method is characterized by having at least twofluorescent dyes mixed within the body of the particle and each one ofthem capable of giving off, simultaneously, multiple fluorescentemission lights of predetermined color and intensity. The combination ofnotions relating to the emission peak corresponding to a given color andintensity of the fluorescent color as expressed in fluorescence channelunits is generally termed as the fluorescence signal. The specific ratioor proportion of dyes at which they are mixed within a population ofparticles will determine the location of said populations on afluorescence map, which allocates these populations according tofluorescent color and brightness. By using as little as two dyes, e.g.,orange and red, as many as 64 populations of beads are made each onedistinct from another by subtle variations in unique fluorescencecharacteristics recognized by a flow cytometry apparatus.

When each such population of beads, characterized by at least twofluorescent signals, is combined with an analytical reactant capable ofbinding a specific analyte in a clinical or test sample a powerfulanalytical tool is obtained, which can provide qualitative andquantitative assay results. The analytical method is also provided whichis based on using multicolored fluorescent beads obtained by the instantinvention. To achieve truly multiplexed analysis of a plurality ofanalytes in a sample, a third type of fluorescent signal, e.g., greenfluorescent signal is provided, usually found in a label reagent, whichis capable of binding the analyte of interest. Thus, methods of makingmulticolored beads, the beads themselves, multiple sets of such beads,and multiplexed methods of analyzing a plurality of analytes in a singlesample are claimed by the instant invention.

A method of staining polymeric microspheres with two or more fluorescentdyes is disclosed, which method comprises: (a) combining at least twofluorescent dyes in a solvent mixture comprising at least one organicsolvent in which the at least two fluorescent dyes are soluble and atleast one alcoholic solvent in which the at least two fluorescent dyesare less soluble, to provide a solution of mixed dyes which is furthercharacterized as having the capacity to swell at least partially but notdissolve a plurality of polymeric microspheres, which is brought intocontact with the solution; (b) contacting a plurality of polymericmicrospheres with the solution for a period of time sufficient toprovide uniform staining of substantially all of the members of theplurality of polymeric microspheres with the at least two fluorescentdyes,

the at least two fluorescent dyes being selected such that on isolationand excitation of the dyed plurality of polymeric microspheres, adistinct fluorescence signal is emitted from each dye, the intensity ofwhich emitted signal is proportional to the amount of the dye in thedyed plurality of polymeric microspheres.

In a particular embodiment of the invention, the method furthercomprises dehydrating the plurality of polymeric microspheres. Such adehydrating step is accomplished by washing the plurality of polymericmicrospheres one or more times with an alcoholic solvent prior tocontacting the microspheres with the solution of mixed dyes. In still apreferred method, the dehydrating step involves drying the washedmicrospheres or allowing the alcoholic solvent to evaporate from thewashed microspheres prior to contacting the microspheres with thesolution of mixed dyes.

Typically, the dyed plurality of polymeric microspheres is isolated byany manner well known in the art, including but not limited tofiltration or centrifugation. It has been found desirable to obtain dyedplurality of polymeric microspheres in which at least one of thefluorescent dyes is diffused throughout the interior of substantiallyall of the members of the dyed plurality of polymeric microspheres, orin which the at least two fluorescent dyes are diffused throughout theinterior of substantially all of the members of the dyed plurality ofpolymeric microspheres. Still other advantages can be gained byproviding dyed plurality of polymeric microspheres in which at least oneof the fluorescent dyes is diffused through only a portion of theinterior of substantially all of the members of the dyed plurality ofpolymeric microspheres.

In a specific method of the invention, the staining procedure furthercomprises preparing a series of the solutions having differing desiredratios of the at least two fluorescent dyes and further comprisescontacting separate populations of a plurality of polymeric microsphereswith the series of the solutions to provide multiple distinctpopulations or subsets of a plurality of polymeric microspheres, eachdistinct population or subset having a differing desired ratio of the atleast two fluorescent dyes.

It has been observed that the distinct fluorescence signals emitted fromthe at least two fluorescent dyes differ in their respective wavelengthsby at least about 10 nm, preferably by at least about 30 nm and mostpreferably by at least about 50 nm.

Hence, the present invention provides a population of polymericmicrospheres substantially uniformly stained with at least twofluorescent dyes, each microsphere of the population upon excitationexhibiting at least two distinct fluorescence emission signalscorresponding to the at least two fluorescent dyes, the intensity ofeach of the at least two emitted signals (i) being proportional to theamount of its corresponding dye in the microsphere, and (ii) exhibitinga coefficient of variation among all the members of the population,which is no greater than about 20 percent. In particular, preferredpopulations are those in which the intensity of each of the at least twoemitted signals exhibits a coefficient of variation among all themembers of the population, which is no greater than about 15 percent,more preferably no greater than about 10 percent and most preferably nogreater than about 8 percent. In still other embodiments, the intensityof each of the at least two emitted signals exhibits a coefficient ofvariation among all the members of the population, which is less thanabout 8 percent.

The present invention offers, thus, a collection of distinct populationsof polymeric microspheres according to the specifications describedabove, each population exhibiting an emission spectrum in a FluorescenceBead Map, which is unique to the population. In specific embodiments,the collection comprises eight or more distinct populations of polymericmicrospheres, preferably sixteen or more distinct populations ofpolymeric microspheres, more preferably twenty-four or more distinctpopulations of polymeric microspheres, most preferably thirty-two ormore distinct populations of polymeric microspheres and still mostpreferably sixty-four or more distinct populations of polymericmicrospheres. Generally, the collection is further characterized in thatthere is substantially no overlap between any of the sixty-four or moreemission spectra associated with the sixty-four or more distinctpopulations of polymeric microspheres.

Also contemplated by the invention, is a method of detectingsimultaneously by flow cytometry a plurality of analytes in a sample,each of the analytes being recognized by a corresponding analyticalreactant, comprising: (a) contacting the sample with a plurality ofpopulations of uniformly stained microspheres, the microspheres havingat least two fluorescent dyes uniformly mixed at a specific ratio withineach microsphere of each the population, each population of themicrospheres having a distinct analytical reactant bound to its surface,wherein, the reactant on each population of microspheres specificallyinteracts with one of the analytes in the sample; (b) providing a labelreagent that specifically binds to the analyte and analyzing themicrospheres to detect the label indicating binding of the analyte tothe analytical reactant; and (c) determining the populations ofmicrospheres having the fluorescent dyes mixed at the specific ratiowithin microspheres of each population to which the reactant is bound.

Other objects of the invention will become apparent from the furtherdiscussions and detailed descriptions provided herein.

OVERVIEW OF THE INVENTION AND ITS EMBODIMENTS

Recent developments in instrumentation have necessitated the concurrentdevelopment of multiple and precisely dyed microspheres that can emitmultiple fluorescent signals simultaneously. This invention describestechniques for absorbing at least two squaric acid-based fluorescentdyes into polymeric, i.e., polystyrene particles.

The present invention describes techniques for precisely dyeingpolystyrene microspheres of sizes ranging from approximately 10 nm to100 μm in diameter. The size of particles is immaterial to thisinvention since the precision of the dyeing process is not affected. Theonly requirement is that particles are made of water-insoluble materialbut soluble in adequate solvents. The dyes employed are preferablysquaric acid-based molecules that exhibit fluorescence extending intonear infrared and/or infrared region, i.e., to ca. 1,000 nm. Use ofother dyes may allow one to expand the range from the ultraviolet toinfrared. This method allows for a highly reproducible process in whichtwo or more dyes of independent concentration are absorbed uniformlyinto each microsphere, resulting in multiple fluorescent signalsrespective of the number of dyes present in the microsphere.

The technology is disclosed enabling one skilled in the art to make aseries of multicolored, fluorescent particles with unique fluorescencecharacteristics and using such particles for multiparameter analysis ofa plurality of analytes simultaneously.

4. BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 showing flow chart with sequential steps of dyeing polymericparticles using the prior art technique.

FIG. 2 showing flow chart with sequential steps of dyeing polymericparticles using the instant invention.

FIG. 3 showing two-dimensional flow cytometry chart illustrating wideoptical distribution of two-color-dyed microspheres using the prior arttechnique.

FIG. 4 showing two-dimensional flow cytometry chart illustrating tightclustering of two-color-dyed microspheres using the instant invention.

FIG. 5 showing that prior art method allows no more than 6 subsets ofmulticolored bead populations on a Fluorescence Bead Map.

FIG. 6 showing Fluorescence Bead Map for 64-Region Bead Set, indicatingtight distribution of each bead subset fluorescence characteristicswithin boundaries prescribed by each region.

5. DETAILED DESCRIPTION OF SPECIFIC EMBODIMENTS

The invention provides novel polymeric beads or microspheres containingat least two fluorescent dyes. This invention further includes theimproved method of making such beads by mixing said beads with at leasttwo fluorescent dyes combined at predetermined ratio so that opticallydistinct, multiple populations of multicolored beads are formed. Thesebead populations are easily discriminated as essentially non-overlappingclusters by visual detection methods such as microscopy or preferably byflow cytometry. The method of simultaneous, multiparameter analysis of aplurality of analytes is also provided whereby each distinctmulticolored bead population would carry an additional analyticalreactant, e.g., antibody, antigen, or nucleic acid probe, which wouldreact with a specific analyte of interest in a sample containing theplurality of analytes.

Polymeric microspheres used in this invention are commercially availablefrom a number of vendors and range in size from 0.01 to 100 micrometers(μm) in diameter. Even though the microparticle can be of any size, thepreferred size is 0.1-50 μm, more preferably 1-20 μm, and even morepreferably 3-9 μm. The sizes of beads in one set can be uniform or maydiffer in order to distinguish and classify them into further subsetsaccording to their size. The size of the microparticle can be measuredin practically any flow cytometry apparatus by so-called forward orsmall-angle scatter light. These subsets can be also furtherdistinguished by different shape of microparticles. The shape of theparticle can be also discriminated by flow cytometry, e.g., byhigh-resolution slit-scanning method.

The preferred make of microspheres is polystyrene or latex material.However, any type of polymeric make of microspheres is acceptableincluding but not limited to brominated polystyrene, polyacrylic acid,polyacrylonitrile, polyacrylamide, polyacrolein, polybutadiene,polydimethylsiloxane, polyisoprene, polyurethane, polyvinylacetate,polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride,polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene,polymethylmethacrylate, or combinations thereof.

The microspheres will also contain 1 to 30% of a cross-linking agent,such as divinyl benzene, ethylene glycol dimethacrylate, trimethylolpropane trimethacrylate, or N,N′methylene-bis-acrylamide or otherfunctionally equivalent agents known in the art. In preferred embodimentmicrospheres are made of polystyrene and contain 1 to 30% divinylbenzene.

The beads may or may not have additional surface functional groups, suchas carboxylates, esters, alcohols, carbamides, aldehydes, amines, sulfuroxides, nitrogen oxides, or halides. The functionality of themicrospheres' surface groups gives the microspheres their couplingcapability allowing chemical binding of analytical reactants. Inaddition to functional groups on microspheres the dyes themselves canalso carry chemically reactive functional groups which in addition togroups listed above can also be carboxylic acid, carboxylic acidsuccinimidyl ester, carboxylic acid anhydride, sulfonyl chloride,sulfonyl fluoride, hydrazine derivatives, acyl azide, isocyanate,haloacetamide, phenols, thiols, and ketones. These functional groups areuseful for attachment of analytical reactants, i.e., classical, commonlyused reactants such as antibody, antigen (hapten), digoxigenin, ornucleic acid probe. These may also include reactants that can formspecific, high-affinity conjugates such as avidin-biotin,receptor-ligand, ligand-ligate, enzyme-substrate, lectin-carbohydrate,protein A-immunoglobulin, etc. For flow cytometry analysis theanalytical reactants are commonly labeled with fluorescent tags orlabels such fluorescein (FITC) or rhodamine. These light-emittingconjugates of a dye and analytical reactant are termed as labelreagents.

The analytical reactants can be also selected among fluorescent reportermolecules capable to react with a variety of analytes, e.g., O₂, CO₂,pH, Ca⁺⁺, Na⁺, K⁺, or Cl⁻ as disclosed for example in U.S. Pat. No.5,747,349 issued to van den Engh et al.

Suitable solvents will be selected based on their ability to solubilizethe particular class of hydrophobic dyes of interest. It is preferablethat their solubility characteristics are substantially similar. Thesolvents can be acyl, aliphatic, cycloaliphatic, aromatic orheterocyclic hydrocarbons; the solvents may or may not have halogens,oxygen, sulfur, nitrogen, and/or phosphorous as either terminal groupsor as integral parts of a ring or chain. Specifically, solvents such astoluene, xylene, hexane, pentane, acetone, DMSO, or methylene chloridecan be used. In a preferred embodiment, chlorinated solvents, morepreferably chloroform, are used to solubilize the squaric acid class ofdyes, which are preferred dyes used in this invention.

In one embodiment two fluorescent squaraine dyes are used, e.g., red dyewhich is1,3-bis[(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydroxy-cyclobutenediylium,bis(inner salt) and orange dye is2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one.The molar ratio between first and second dye, when present in a bead,will preferably be between about 0 and 10,000, more preferably between0.00001 and 2,000. Both dyes would preferably be excited at the sameabsorption wavelength, e.g., ranging from ultraviolet to about 800 nm,and emit fluorescent light at two distinct, essentially non-overlappingwavelengths distant from each other by at least 10 nm, preferably 30 nm,and more preferably by at least 50 nm. For example, the emission peak ofthe dye #1 is at 585 nm, and the peak emission of dye #2 is at 630 nm.

The squaric acid based fluorescent dyes can be synthesized by methodsdescribed in the literature. See, for example, Sprenger et al. Angew.Chem., 79, 581 (1967); Angew. Chem. 80, 541 (1968); and Maaks et al.,Angew Chem. Intern. Edit. 5, 888 (1966). Briefly, one equivalent ofsquaric acid (1,2-dihydroxycyclobutenedione) is condensed with twoequivalents of an active compound, such as a pyrrole, indoline, oraniline, and refluxed in a mixture of an alcohol and an aromatic solvent(such as benzene) under conditions that allow removal of water from thereaction mixture. The resulting dye can be collected and purified by anumber of standard methods, such as recrystallization, distillation,chromatography, etc. Additionally, unsymmetrically substituted squaricacid compounds can be synthesized by methods such as those described byLaw et al., J. Org. Chem. 57, 3278, (1992). Specific methods of makingsome of such dyes are well known in the art and can be found for examplein U.S. Pat. Nos. 5,795,981; 5,656,750; 5,492,795; 4,677,045; 5,237,498;and 5,354,873. Optionally such dyes will contain functional groupscapable of forming a stable fluorescent product with functional groupstypically found in biomolecules or polymers including activated esters,isothiocyanates, amines, hydrazines, halides, acids, azides, maleimides,alcohols, acrylamides, haloacetamides, phenols, thiols, acids, aldehydesand ketones.

In addition to specific squaric acid dyes are used in this preferredembodiment, related dyes can be further selected from cyclobutenedionederivatives, substituted cephalosporin compounds, fluorinated squarainecompositions, symmetrical and unsymmetrical squaraines, alkylalkoxysquaraines, or squarylium compounds. Some of these dyes can fluoresce atnear infrared as well as at infrared wavelengths that would effectivelyexpand the range of emission spectra up to about 1,000 nm.

In addition to squaraines, i.e., derived from squaric acid, hydrophobicdyes such as phthalocyanines and naphthalocyanines can be also selectedas operating at longer wavelengths. Other classes of fluorochromes areequally suitable for use as dyes according to the present invention.Some of these dyes are listed herein: 3-Hydroxypyrene 5,8,10-TriSulfonic acid, 5-Hydroxy Tryptamine, 5-Hydroxy Tryptamine (5-HT), AcidFuhsin, Acridine Orange, Acridine Red, Acridine Yellow, Acriflavin, AFA(Acriflavin Feulgen SITSA), Alizarin Complexon, Alizarin Red,Allophycocyanin, ACMA, Aminoactinomycin D, Aminocoumarin, AnthroylStearate, Aryl- or Heteroaryl-substituted Polyolefin, Astrazon BrilliantRed 4G, Astrazon Orange R, Astrazon Red 6B, Astrazon Yellow 7 GLL,Atabrine, Auramine, Aurophosphine, Aurophosphine G, BAO 9(Bisaminophenyloxadiazole), BCECF, Berberine Sulphate, Bisbenzamide,BOBO 1, Blancophor FFG Solution, Blancophor SV, Bodipy FI, BOPRO1,Brilliant Sulphoflavin FF, Calcien Blue, Calcium Green, Calcofluor RWSolution, Calcofluor White, Calcophor White ABT Solution, CalcophorWhite Standard Solution, Carbocyanine, Carbostyryl, Cascade Blue,Cascade Yellow, Catecholamine, Chinacrine, Coriphosphine O, Coumarin,Coumarin-Phalloidin, CY3.1 8, CY5.1 8, CY7, Dans (1-Dimethyl AminoNaphaline 5 Sulphonic Acid), Dansa (Diamino Naphtyl Sulphonic Acid),Dansyl NH—CH3, DAPI, Diamino Phenyl Oxydiazole (DAO),Dimethylamino-5-Sulphonic acid, Dipyrrometheneboron Difluoride, DiphenylBrilliant Flavine 7GFF, Dopamine, Eosin, Erythrosin ITC, EthidiumBromide, Euchrysin, FIF (Formaldehyde Induced Fluorescence), FlazoOrange, Fluo 3, Fluorescamine, Fura-2, Genacryl Brilliant Red B,Genacryl Brilliant Yellow 10GF, Genacryl Pink 3G, Genacryl Yellow 5GF,Gloxalic Acid, Granular Blue, Haematoporphyrin, Hoechst 33258 (bound toDNA), Indo-1, Intrawhite Cf Liquid, Leucophor PAF, Leucophor SF,Leucophor WS, Lissamine Rhodamine B200 (RD200), Lucifer Yellow CH,Lucifer Yellow VS, Magdala Red, Marina Blue, Maxilon Brilliant Flavin 10GFF, Maxilon Brilliant Flavin 8 GFF, MPS (Methyl Green PyronineStilbene), Mithramycin, NBD Amine, Nile Red, Nitrobenzoxadidole,Noradrenaline, Nuclear Fast Red, Nuclear Yellow, Nylosan BrilliantFlavin E8G, Oregon Green, Oxazine, Oxazole, Oxadiazole, Pacific Blue,Pararosaniline (Feulgen), Phorwite AR Solution, Phorwite BKL, PhorwiteRev, Phorwite RPA, Phosphine 3R, Phthalocyanine, Phycoerythrin R,Polyazaindacene Pontochrome Blue Black, Porphyrin, Primuline, ProcionYellow, Propidium Iodide, Pyronine, Pyronine B, Pyrozal Brilliant Flavin7GF, Quinacrine Mustard, Rhodamine 123, Rhodamine 5 GLD, Rhodamine 6G,Rhodamine B, Rhodamine B 200, Rhodamine B Extra, Rhodamine BB, RhodamineBG, Rhodamine WT, Rose Bengal, Serotonin, Sevron Brilliant Red 2B,Sevron Brilliant Red 4G, Sevron Brilliant Red B, Sevron Orange, SevronYellow L, SITS (Primuline), SITS (Stilbene Isothiosulphonic acid),Stilbene, Snarf 1, sulphO Rhodamine B Can C, Sulpho Rhodamine G Extra,Tetracycline, Texas Red, Thiazine Red R, Thioflavin S, Thioflavin TCN,Thioflavin 5, Thiolyte, Thiozol Orange, Tinopol CBS, TOTO 1, TOTO 3,True Blue, Ultralite, Uranine B, Uvitex SFC, Xylene Orange, XRITC, YOPRO 1, or combinations thereof. One skilled in the art would certainlyknow which one to select among such dyes as long as desired emission andabsorption properties as well as their hydrophobic properties areappropriate. The spectral properties of the fluorescent dyes should besufficiently similar in excitation wavelengths and intensity tofluorescein or rhodamine derivatives as to permit the use of the sameflow cytometry equipment. It is preferable that the dyes, however, havehigher solubility in organic solvents and have improved photostabilityand quantum yields. These dyes will be combined at predetermined ratioand embedded into a microsphere vehicle and total dye quantity will bebetween about 0.00001% and 15% by weight to particle weight. Thislimitation is however of little consequence to the present invention foras long as the particle impregnated with said dyes is stable and usablefor its intended purpose.

Prior Art Method.

The prior art method teaches staining carrier particles with a singledye only (see FIG. 1). However, for the purpose of a meaningfulcomparison and in order to be consistent with the thrust of the instantinvention, the said method was adapted to stain with two dyessimultaneously. The prior art of dyeing large polymer particles (>5 um)as stated in “Uniform Latex Particles” by Leigh B. Bangs was performedand the general outline of the procedure is shown in FIG. 1 and obtainedresults are shown in FIG. 3. Briefly, the process is started by placing5 ml of undyed stock microspheres in an aqueous medium directly on amembrane covered fritted funnel. A vacuum pump pulled air through themicrospheres plated onto the filter paper for one hour. Next, the driedmicrospheres were transferred to 50 ml of dye solution, covered, andstirred at room temperature over night. The next day the microsphereswere separated by filtration from the dye solution and the dyedparticles were placed in a vacuum dessicator for about four hours toremove residual solvent. Next is added 200 ml of Triton X-100 and watersolution to the dried dyed microspheres in a 250 ml flask. The solutionis stirred for three hours. The solution is filtered and the washing isrepeated until no further dye is detected in the filtrate. The beadsstained in this manner are tested for staining uniformity by flowcytometry (FIG. 3). It can be easily seen that, based on three separateexperiments (tests A, B, and C), bead-to-bead variation at FL2 and FL3parameters (CV or coefficient of variation) is rather high andinadequate (Table 1) to satisfy the increasing demand for precisely dyedmulticolored microspheres.

General Outline of the Instant Method.

As the demand and applications for precisely dyed multicoloredmicrospheres increases the development of alternate processes to theaforementioned method is warranted. As a result, a modification of theprior art method has been developed which has proven to be the mostefficient method for precision dyeing of the microspheres (see FIG. 2).This method takes one tenth or even less of the time of the previouslymentioned method and significantly enhances its precision. As before, itis critical to remove almost all traces of water from the microspheres.To accomplish this a volume of stock microspheres in an aqueous mediumis pipetted onto a vacuum filter membrane, and the liquid is removed anddiscarded. Next, 100 ml of the rinse solvent (an aliphatic alcohol, suchas propanol, methanol, ethanol, etc.) are added to the microspheres. Themicrospheres are resuspended by placing an ultrasonic probe directlyinto the solution and applying power for several seconds or as needed toaffect resuspension. The suspension is filtered and previous step isrepeated once more. Dyeing of the microspheres is accomplished by adding50 ml of a dye solution (composed of one or more dyes in an organicsolvent, as described below) to the filtering cup and resuspending asbefore. The suspension is allowed to sit for five minutes in thefiltering cup. Next, 50 ml of rinse solvent is added to the dyesuspension, sonicated and filtered. Another 100 ml of the rinse solventis added, resuspended and filtered. The last step is repeated once more.In order to prepare the microspheres for storage, 100 ml of an aqueousmedium is added to the microspheres, then sonicated and filtered.Finally, 50 ml of aqueous medium is added to the microspheres, sonicatedand transferred to a storage container.

In a particular embodiment of the invention, two squaric acid-based dyesare mixed in a solvent suitable for the complete dissolution of bothdyes, such as chloroform. Ethanol is added to the solution to increasewetting of the microspheres, and to create a process-dependent, finalsolvent density that is less than that of the microspheres. Theconcentrations of each dye are experimentally determined as a functionof the target fluorescence intensity at each of the two centerwavelengths. These concentrations maintain their relative intensitythroughout this inventive process.

An important aspect of the present invention is the preparation ofmicrospheres prior to the dyeing operation. Manufacturers often supplymicrospheres in an aqueous medium. It has been discovered that thesurface of the microsphere that had been stored in aqueous medium mustbe treated to make it permeable to organic compounds. In a preferredembodiment, an amount of a polar organic solvent such as an alcohol isadded to the microsphere solution to achieve about a 50% mixture of theaqueous medium and the polar organic solvent. This ratio, however, mayvary and adjusted at will according to particular needs that one mayhave or determined by chemical and physical properties of medium andsolvent.

An equally efficient and precise technique involves “drying” themicrospheres through a series of alcohol, e.g., methanol, ethanol,2-propanol, rinses. The process begins by spinning down the aqueoussuspension of microspheres, typically 10% solids in suspension. Theaqueous medium is decanted, and the beads are re-suspended in methanol.The alcohol solution at ca. 5% solids is vortexed, sonicated and spundown. This step is performed once or twice more. The excess alcohol isdecanted from the pellet, and residual solvent is evaporated undervacuum.

Test samples consisting of 0.05 gram of dried microspheres are used tohelp adjust the dye solution to its desired ratio. The dried 0.05 gramof microspheres are suspended in 0.5 ml of dye mixture containing two ormore dyes of interest. The suspension of microspheres, now at 10%solids, is vortexed and sonicated into suspension. Once in suspensionthe mixture of microspheres and dyes is mixed for one hour. After thathour, the microspheres are spun down for a period of 1 minute using acentrifuge. The dye solution is decanted back into the main flask, andthe 0.05 g of microspheres are re-suspended in 1 ml of 90% of alcohol,e.g., methanol. The rinse step uses double the volume of the dyesolution, thus maintaining a 5% solid solution. The sample is vortexed,sonicated and spun down. The methanol supernatant is decanted. The 90%methanol rinse step is repeated once more. Finally, the excess methanolis decanted from the pellet, and the microspheres are re-suspended in anaqueous medium. The resulting test samples are then tested to determinethe fluorescence activity/intensity of the labeled beads.

When the test samples show that the dye solution, indeed, has theprecise ratios of the desired dyes, a macro-scale batch is conducted.The principle of macro-scale work up is identical to that noted above.Briefly, 25 ml of the desired dye solution is transferred to a 50 mlvial, which contains 2.5 grams of dried microspheres. The microspheres,now at 10% solids, are vortexed and sonicated. Once the microspheres arecompletely in suspension, it is mixed for an hour. After that hour themicrospheres are taken out of the dye solution by centrifugation. Thedye solution is decanted back into the main flask, and the 2.5 grams ofmicrospheres are re-suspended in 50 ml of 90% methanol. The rinse stepuses double the volume of the dye solution, thus maintaining a mixtureof 5% solids. The sample is vortexed, sonicated and spun down. Themethanol supernatant is decanted. This step is repeated once more. Afterthe final methanol rinse is decanted, the microspheres are put throughan aqueous rinse. The aqueous supernatant is decanted, and the beads arethen re-suspended and stored in a fresh aqueous medium.

The following Examples are presented to illustrate the advantages of thepresent invention and to assist one of ordinary skill in making andusing the same. These Examples are not intended in any way to otherwiselimit the scope of the disclosure or the protection granted by LettersPatent hereon.

EXAMPLE 1

A single solution containing two different squaric acid dyes isprepared. One dye is a red fluorescent dye1,3-bis[(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydroxy-cyclobutenediylium,bis(inner salt) and second dye is orange fluorescent dye can be2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one.The peak emission of dye #1 is 585 nm, and the peak emission of dye #2is 630 nm. These dyes are chosen because they fall in the center of twoof the fluorescence channels of a Becton Dickinson FACScan flowcytometer, which is the measurement device used to determine theprecision of prior art dyeing techniques compared with this innovativenew technique. The choice of fluorescence channels is, however, relativeand immaterial since another flow cytometry apparatus may have differentsettings.

Two samples of undyed microspheres are prepared. The first is dyed withthe mixture of orange and red dyes using this innovative technique(shown FIGS. 2 and 4), and the second is dyed using the prior arttechnique (FIGS. 1 and 3). Samples are measured on the FACScan, and anX-Y plot is generated to show the relative homogeneity of each sample.X-axis represents brightness or fluorescence intensity of orange dye andY-axis represents the same parameters of red dye. Mean intensities andcoefficients of variation are also measured. It is clear that beadsstained by the old method spread over much larger X-Y area, indicatingthat the ratio of orange and red dyes vary from particle-to-particle. Incontrast, the coefficient of variation in the bead population dyed bythe instant, improved method is much smaller. About 10,000 beads in eachtests A, B, and C, were run in parallel with beads stained by Bangsmethod (Table 1).

EXAMPLE 2

To make another population of beads with different fluorescentcharacteristics the ratio of red/orange dyes is altered by an adequateincrement in proportion of dyes so that obtained population opticallydoes not overlap with the former population. The prior art failed toprovide multiple populations of multicolored beads due to inevitableintra-sample heterogeneity resulting from inadequate staining processresulting in poor dye distribution from particle-to-particle withingiven staining batch. Thus, upon excitation with a light source, stainedbeads containing more than one dye failed to emit uniform fluorescencesignals of desired intensity. The instant invention overcomes thisproblem and achieves construction of as many as 64 subsets of opticallydistinct beads by varying the ratio of just 2 dyes. This example is notin any way a limiting one since one of ordinary skill may easilygenerate smaller or higher number of bead subsets by using the instantteaching. One skilled in the art may appreciate that nothing even closeto this achievement has ever been enabled in the actual practice.Although such an eventuality was theoretically speculated as a possibleone, the prior art failed to teach one of ordinary skill how to arriveat that.

The present inventors were able, for the first time, to reduce topractice the invention and representative experimental results ofobtaining 64-bead population are shown in FIG. 6 and Table 2. Theresults illustrated in FIG. 5 show multicolored beads by using stainingprocedure of the prior art method. Due to imprecision in stainingtechnique, which results in a wide dispersion of dyes ratio frombead-to-bead, no more than 6 subsets of multicolored bead populationscan be fitted on a Fluorescence Bead Map. In contrast, FIG. 6 showsFluorescence Bead Map containing 64 populations of beads, indicatingtight distribution of each bead subset fluorescence characteristicswithin boundaries prescribed by each region. The cross-talk betweenvarious clusters is minimal. Most of the overlap is due to the presenceof bead agglomerations which emit brighter signal but they areeliminated by size discrimination based on light scatter.

In general, as can be readily glanced from Table 2, there is anunequivocal relationship between two dye concentrations in a givenpopulation of beads and location of said populations on X-Y map. Eachlocation is assigned in terms of red (FL3) or orange (FL2) dyesintensity as expressed in linear fluorescence channels units which fallin approximately 470, 580, 690, 750, 800, 900, and 990 series. Forpractical reasons, i.e., space limitation in the bead cluster, the lastdigit “0” is omitted. The first two digits in each bead populationrepresents fluorescence intensity of orange dye (FL2) and last twodigits the intensity of the red dye (FL2). The fluorescent intensityincreases as the numbers go higher. The beads with lowest intensity(4725) reside in lower left corner and brightest ones (9998) in upperright corner. As they move vertically up a column, both red and orangedye amounts in a bead must be increased. This is because there is asubstantial amount of energy transfer from the orange dye to the red.When moving horizontally from left to right across a row, the red dyemust be decreased in order to maintain a steady FL3 value. This is dueto overlap of the orange dye spectrum into the red region, thusnecessitating the increase in FL3 signal. In this manner multiple,non-overlapping populations of beads are constructed. Two parametersnamely, a fluorescent color (red or orange) and color intensity orbrightness (expressed in fluorescence channel units), are essential toclassify obtained beads and are termed as a fluorescence signal.

Hence, particular populations of beads are provided whose fluorescencecharacteristics or signals fall within a prescribed region depicted in aFluorescence Bead Map. Typically, about 80% or greater of the individualbeads within a particular population of beads will exhibit fluorescencecharacteristics within the desired region, preferably about 90% orgreater, more preferably about 97% or greater, most preferably about 99%or greater. For each set of beads, typically about 1% or less of theindividual beads within a particular set of beads will exhibitfluorescence characteristics that fall within another, undesired region,preferably about 0.5% or less, more preferably about 0.3% or less, mostpreferably about 0.2% or less.

While theoretically any number of populations or subsets can be presentin each Bead Map, due to the limitations in the prior art techniques itis not possible to obtain more than 6 subsets coexisting simultaneously.While theoretically it has been speculated that such subsets can beextremely valuable for multiplex analysis (see for example McHugh, “FlowMicrosphere Immunoassay for the Quantitative and Simultaneous Detectionof Multiple Soluble Analytes,” in Methods in Cell Biology, 42, Part B,(Academic Press, 1994) so far there are no known examples in the artenabling and demonstrating the reduction to practice of tangible,multicolored beads. At best only 1 and perhaps a maximum of 5 populationof beads containing various ratios of two dyes could have been possible.For example, U.S. Pat. No. 4,717,655 discloses such beads, however, thedisclosure was not enabled and the method of incorporating dyes in thesebeads is by copolymerization process and as such it is unrelated to theinstant invention. In contrast, due to a significant improvement overexisting methodology it is now technically possible to obtain 16-subset,32-subset, 64-subset or even higher number of bead collections using theinstant methodology.

As an example 64-subset bead collection or 64 populations of beads wereconstructed each population differing from another by a distinctlocation on the X-Y plot. These locations essentially do not overlap. Asopposed to the prior art methods which result in up to 10-20% or evenhigher rate of dispersion the instant method allows to obtainessentially homogeneous populations of beads with only 0.2-0.3%dispersion. As used hereinafter the term essentially non-overlapingpopulations means that only about 0.2-0.3% of beads in each populationmay display an optical pattern or fluorescent signal which can beascribed to the neighboring cluster of beads having the same set offluorescent dyes but mixed at different ratio. This is a significantimprovement over the prior art.

EXAMPLE 3

Although multiplexed analysis capability theoretically would provideenormous benefit in the art of flow cytometry, very little progress hasbeen previously achieved due to technical limitations in obtainingsufficient variety of multicolored, non-overlaping subsets offluorescent beads. A review of some of these prior art techniques isprovided by McHugh (see above). These methods have been unsatisfactoryas applied to provide fully multiplexed assay capable of analysis ofmore than a few different analytes. In the prior art when beads wereincorporating a combination of 2 dyes only 5 subsets of beads wereallegedly obtained (U.S. Pat. No. 4,717,655 issued to Fulwyler). A setwith maximum of six subsets is obtained using Bangs method (seeExample 1) which is still insufficient for the purposes of trulymultiplexed assay.

A series of antibodies, antigens, or nucleic acid probes, collectivelynamed hereinafter as analytical reactants, are attached to the beads byany of a number of conventional procedures such as by chemical orphysical adsorption as described by Colvin et al., “The Covalent Bindingof Enzymes and Immunoglobulins to Hydrophilic Microspheres” inMicrospheres: Medical and Biological Applications, 1-13, CRC, BocaRaton, Fla., 1988; Cantarero et al., “The Adsorptive Characteristics ofProteins for Polystyrene and Their Significance in Solid-PhaseImmunoassays,” Anal Biochem, 105, 375-382 (1980); and Illum et al.,“Attachment of Monoclonal Antibodies to Microspheres,” Methods inEnzymol, 112, 67-84 (1985) 112, 67-84 (1985).

After attachment of a reactant to the beads' surface, aliquots from eachsubset are mixed to create a pool containing known amounts of beadswithin each subset. Preferably, the pooled set is prepared with equalvolumes of beads from each subset, so that the set contains about thesame number of beads from each subset or population. This pool is thenbe incubated with a fluid sample of interest, such as serum or plasma,to test for the presence of antibodies in the fluid that are reactivewith antigens on the beads. Such incubation is generally performed underconditions of temperature, pH, ionic concentrations, and the like thatfacilitate specific reaction of antibodies in the fluid sample withantigen on the bead surface. After a sufficient period of time, thebeads in the mixture are centrifuged, washed and incubated for anotherperiod of time with a “secondary” antibody such as, for example,fluorescein labeled goat anti human immunoglobulin. The secondaryantibody or label reagent will bind to and fluorescently labelantibodies bound to antigen on the beads. After washing (or withoutwashing), the beads are processed by a flow cytometer and the fourclassification parameters forward light scatter, side light scatter, redfluorescence, and orange fluorescence are measured and used to identifythe subset or population to which each bead belongs. A simultaneousmeasurement of green fluorescence (measurement parameter) for each beadallows one to determine whether the bead has antibody bound to it.Because the subset to which a bead belongs is correlated with thepresence of a particular antigen, e.g., series of grass allergens,various substance abuse drugs, one may readily determine the specificityof the antibody bound to a bead as a function of the subset to which itbelongs.

Displacement or Competition Assay

Assays for many substances in a clinical laboratory are based on theinterference with specific ligand-ligate or antigen-antibodyinteractions. In these assays, one member of the ligand-ligate pair islabeled with the fluorophore or fluorochrome and one member isimmobilized on the beads. Soluble, unlabeled analyte, which may beligand or ligate, is added to the reaction mixture to competitivelyinhibit interaction of the labeled component with the immobilizedcomponent. It is usually not important which member of the pair islabeled and which is immobilized; however, in certain assays, functionaladvantages may dictate the orientation of the assay. In an exemplaryassay of this type, each bead subset is provided with an antigen. Theantigen-coated beads are then reacted with labeled antibody specific forthe antigen on the bead surface. Subsequent addition of a test fluidcontaining soluble analyte (inhibitor) will displace the labeledantibody from the beads in direct proportion to the concentration of thesoluble analyte. A standard curve of known analyte concentrations isused to provide accurate quantification of analyte in the test sample.

Nucleic Acid Analysis

The power and sensitivity of PCR found its application to a wide varietyof analytical problems in which detection of DNA or RNA oligonucleotidesequences is required. One major difficulty with the PCR technique isthe cumbersome nature of the methods of measuring end-product, i.e.,amplified DNA. A flow cytometric bead-based hybridization assay permitsthe extremely rapid and accurate detection of genetic sequences ofinterest. In a preferred embodiment of this invention, a bead to which anucleic acid segment of interest has been coupled is provided. A PCRproduct of interest (or any other DNA or cDNA segment) is detected byvirtue of its ability to competitively inhibit hybridization between thenucleic acid segment on the bead and a complementary fluorescent DNAprobe. The method is sensitive and precise and allows the detection ofsingle point mutations in the PCR product or DNA of interest. Themultiplexed DNA analysis method can be applied to detect any PCR productor other DNA of interest for specific polymorphisms or mutations and oneskilled in the art will recognize that numerous applications can beimagined such as presence of histocompatibility alleles associated withsusceptibility to diseases, mutations associated with genetic diseases,autoimmune diseases, or mutations of oncogenes associated with neoplasiaor risk of neoplasia. In a same way nucleic acid segments frompathogenic organisms such as bacterial, viral, fungal, mycoplasmal,rickettsial, chlamydial, or protozoan pathogens can be detectedsimultaneously.

Enzyme Assays

The invention is also useful for measurement of enzymes, enzymeinhibitors and other analytes. For example, bead subsets are generatedwith selected fluorescent substrates which are enzymatically cleavedfrom the bead, resulting in a loss of fluorescence. Enzymes that can bedetected and measured using the invention include but are not restrictedto, proteases, glycosidases, nucleotidases, and oxidoreductases. Anyenzyme that results in selected bond cleavage can be measured.Alternatively, the action of the enzyme on the bead-bound substrateresults in the formation or identification of a ligate for a fluorescentligand present in the reaction mixture. The bead bearing the modifiedsubstrate then becomes fluorescent by virtue of binding of thefluorescent ligand to the newly formed ligate. Because each type of beadbearing the unique substrate can be distinguished, a mixture of beadsubsets can be used to measure several enzyme activities simultaneouslyin the same reaction mixture.

Fluids or samples with analytes that can be analyzed using thesetechniques include plasma, serum, tears, mucus, saliva, urine, pleuralfluid, spinal fluid and gastric fluid, sweat, semen, vaginal secretions,fluid from ulcers and other surface eruptions, blisters, and abscesses,and extracts of tissues including biopsies of normal, malignant, andsuspect tissues.

The above examples can be used to perform most common immunodiagnosticand nucleic acid assays. Other applications such as high throughputscreening of combinatorial chemistry libraries for discovering newdrugs, environmental screening of pollutants, drug testing, foodsafety-related investigations, testing of multiple analytes foragricultural needs, etc, can be imagined.

It is to be understood that, while the foregoing invention has beendescribed in detail by way of illustration and example of preferredembodiments, numerous modifications, substitutions, and alterations arepossible without departing from the spirit and scope of the invention asdescribed in the following claims.

TABLE 1 Sample FL2 (CV) FL3 (CV) Events Old Method A 24.2 25.9 10000 B27.7 25.5 10000 C 27.7 23.2 10000 Improved Method A 7.3 6.8 10000 B 7.36.7 10000 C 7.2 6.7 10000

TABLE 2 FL3 Red Dye (mM) FL2 Orange Dye (mM) Region 47 series 470/2500.0495 0.00441 470/340 0.104 0.0041 470/415 0.0685 0.004 470/470 0.3880.00576 470/525 0.458 0.0039 470/580 0.981 0.00662 470/635 0.132 0.00557470/690 1.831 0.00637 470/745 3.84 0.01138 470/810 11.29 0.0278 Region58-series 580/250 0.0201 0.00994 580/340 0.0582 0.00979 580/415 0.07630.00161 580/470 1.38 0.0256 580/525 0.42 0.0114 580/580 0.613 0.0162580/635 1.15 0.0132 580/690 2.71 0.0267 580/745 0.643 0.0248 580/810 7.50.0489 580/870 9.71 0.008 Region 69-series 690/360 0.048 0.024 690/4150.129 0.024 690/470 0.226 0.023 690/525 0.45 0.029 690/580 0.89 0.0459690/635 1.11 0.0322 690/690 2.45 0.07 690/745 2.88 0.0624 690/810 5.540.185 690/870 5.68 0.145 750/940 10.52 1.07 Region 80-series 800/4150.0465 0.15 800/470 0.123 0.131 800/525 0.226 0.061 800/580 0.504 0.0835800/635 0.62 0.0805 800/690 1.18 0.118 800/745 1.7 0.113 800/800 3.060.218 800/870 5.7 0.31 800/940 7.54 0.806 950/940 7.93 0.736 Region90-series 900/510 0.084 0.0017 900/553 0.0666 0.0146 900/596 0.1380.0913 900/645 1.29 0.253 900/690 0.861 0.298 900/734 0.699 0.229900/779 0.984 0.214 900/823 1.13 0.435 900/867 2.61 0.381 900/912 3.410.579 900/953 4.88 0.861 Region 99-series 990/600 — 0.274 990/640 0.1250.21 990/680 0.21 0.268 990/720 0.472 0.216 990/760 0.712 0.275 990/8000.82 0.278 990/840 1.12 0.446 990/880 1.87 0.431 990/920 3.24 0.477990/980 2.93 0.763

1. A population of polymeric microspheres dyed with two or morefluorescent dyes, obtained by a method comprising: treating polymericmicrospheres by adding a polar organic solvent to an aqueous solutioncontaining the polymeric microspheres to produce a mixture; and addingat least two fluorescent dyes to the mixture such that the polymericmicrospheres are stained with the at least two fluorescent dyes, whereinthe at least two fluorescent dyes are selected such that on isolationand excitation of the polymeric microspheres, a distinct fluorescencesignal is emitted from each dye, wherein the distinct fluorescencesignals have an intensity proportional to the amount of the dye in thepolymeric microspheres, and wherein the distinct fluorescence signalsare substantially equivalent to targeted fluorescence signals that havebeen used to determine a concentration ratio of the at least twofluorescent dyes added to the mixture.
 2. The population of claim 1,wherein the polar organic solvent is an aliphatic alcohol, methanol,ethanol, or propanol.
 3. The population of claim 1, wherein theintensity exhibits a coefficient of variation among all of the polymericmicrospheres that is no greater than about 15 percent.
 4. The populationof claim 1, wherein the intensity exhibits a coefficient of variationamong all of the polymeric microspheres that is no greater than about 10percent.
 5. The population of claim 1, wherein the intensity exhibits acoefficient of variation among all of the polymeric microspheres that isno greater than about 8 percent.
 6. The population of claim 1, whereinthe at least two fluorescent dyes are hydrophobic.
 7. The population ofclaim 1, wherein the at least two fluorescent dyes comprise squaricacid-based dyes.
 8. The population of claim 7, wherein the squaricacid-based dyes are cyclobutenedione derivatives, symmetrical andunsymmetrical squaraines, substituted cephalosporin compounds,fluorinated squaraine compositions, alkylalkoxy squaraines, orsquarylium compounds.
 9. The population of claim 7, wherein the squaricacid-based dyes are a red fluorescent dye or an orange fluorescent dye.10. The population of claim 9, wherein the red fluorescent dye comprises1,3-bis[(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)methyl]-2,4-dihydroxycyclobutenediylium,bis(inner salt), and wherein the orange fluorescent dye comprises2-(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one.11. The population of claim 1, wherein the polymeric microspherescomprise polystyrene, brominated polystyrene, polyacrylic acid,polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane,polybutadiene, polyisoprene, polyurethane, polyvinyl acetate,polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride,polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene,polyglycidylmethacrylate, polymethylmethacrylate, or copolymers, blends,composites, or combination thereof.
 12. The population of claim 1,wherein the polymeric microspheres comprise at least one analyticalreactant bound covalently to functional groups present on a surface ofthe polymeric microspheres or passively adsorbed to the surface of thepolymeric microspheres.
 13. The population of claim 1, wherein thepolymeric microspheres have a diameter between about 10 nm and 100 μm.14. A collection of distinct populations of polymeric microspheresaccording to claim 1, each population exhibiting an emission spectrum ina Fluorescence Bead Map, which is unique to said population.
 15. Thecollection of claim 14, comprising eight or more distinct populations ofthe polymeric microspheres.
 16. The collection of claim 14, comprisingsixteen or more distinct populations of the polymeric microspheres. 17.The collection of claim 14, comprising twenty-four or more distinctpopulations of the polymeric microspheres.
 18. The collection of claim14, comprising thirty-two or more distinct populations of the polymericmicrospheres.
 19. The collection of claim 14, comprising sixty-four ormore distinct populations of the polymeric microspheres.
 20. Thecollection of claim 19, wherein there is substantially no overlapbetween any of the sixty-four or more emission spectra associated withthe sixty-four or more distinct populations.
 21. A set of distinctpopulations of polymeric microspheres dyed with at least two fluorescentdyes, wherein the set of distinct populations of polymeric microspheresis dyed using a method comprising: treating separate populations ofpolymeric microspheres by adding a polar organic solvent to aqueoussolutions containing the separate populations of polymeric microspheresto produce different mixtures; preparing a series of solutions, eachcomprising at least two fluorescent dyes in a solvent, wherein each ofthe solutions in the series has a different concentration of at leastone of the at least two fluorescent dyes; and adding said each of thesolutions to one of the different mixtures to provide the set ofdistinct populations of polymeric microspheres, wherein each of thedistinct populations of polymeric microspheres emits a differenttargeted fluorescence intensity from the at least one of the at leasttwo fluorescent dyes upon excitation, and wherein the different targetedfluorescence intensities have been used to determine the differentconcentrations of the at least two fluorescent dyes included within eachof the solutions.
 22. The set of claim 21, wherein the polar organicsolvent is an aliphatic alcohol, methanol, ethanol, or propanol.
 23. Theset of claim 21, wherein the different fluorescence intensity of saideach of the distinct populations exhibits a coefficient of variation ofno greater than about 15 percent.
 24. The set of claim 21, wherein thedifferent fluorescence intensity of said each of the distinctpopulations exhibits a coefficient of variation of no greater than about10 percent.
 25. The set of claim 21, wherein the different fluorescenceintensity of said each of the distinct populations exhibits acoefficient of variation of less than about 8 percent.
 26. The set ofclaim 21, wherein the at least two fluorescent dyes are hydrophobic. 27.The set of claim 26, wherein the at least two fluorescent dyes comprisesquaric acid-based dyes.
 28. The set of claim 27, wherein the squaricacid-based dyes comprise cyclobutenedione derivatives, symmetrical andunsymmetrical squaraines, substituted cephalosporin compounds,fluorinated squaraine compositions, alkylalkoxy squaraines, orsquarylium compounds.
 29. The set of claim 27, wherein the squaricacid-based dyes comprise a red fluorescent dye or an orange fluorescentdye.
 30. The set of claim 29, wherein the red fluorescent dye comprises1,3-bis[(1,3-dihydro-1,3,3-trimethyl2H-indol-2-ylidene)methyl]-2,4-dihydroxycyclobutenediylium,bis(inner salt), and wherein the orange fluorescent dye comprises2(3,5-dimethylpyrrol-2-yl)-4-(3,5-dimethyl-2H-pyrrol-2-ylidene)-3-hydroxy-2-cyclobuten-1-one.31. The set of claim 21, wherein the polymeric microspheres comprisepolystyrene, brominated polystyrene, polyacrylic acid,polyacrylonitrile, polyacrylamide, polyacrolein, polydimethylsiloxane,polybutadiene, polyisoprene, polyurethane, polyvinylacetate,polyvinylchloride, polyvinylpyridine, polyvinylbenzylchloride,polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene,polyglycidylmethacrylate, polymethylmethacrylate, or copolymers, blends,composites, or combinations thereof.
 32. The set of claim 21, whereinthe polymeric microspheres comprise at least one analytical reactantbound covalently to functional groups present on a surface of thepolymeric microspheres or passively adsorbed to the surface of thepolymeric microspheres.
 33. The set of claim 21, wherein the polymericmicrospheres have a diameter between about 10 nm and 100 μm.
 34. The setof claim 21, wherein said each of the distinct populations exhibits anemission spectrum in a Fluorescence Bead Map that is unique to said eachof the distinct populations.
 35. The set of claim 21, comprising eightor more of the distinct populations of polymeric microspheres.
 36. Theset of claim 21, comprising sixteen or more of the distinct populationsof polymeric microspheres.
 37. The set of claim 21, comprisingtwenty-four or more of the distinct populations of polymericmicrospheres.
 38. The set of claim 21, comprising thirty-two or more ofthe distinct populations of polymeric microspheres.
 39. The set of claim21, comprising sixty-four or more of the distinct populations ofpolymeric microspheres.
 40. The set of claim 39, wherein said each ofthe distinct populations exhibits an emission spectrum in a FluorescenceBead Map that is unique to said each of the distinct populations, andwherein there is substantially no overlap between any of the sixty-fouror more emission spectra associated with the sixty-four or more of thedistinct populations of polymeric microspheres.
 41. A population ofpolymeric microspheres dyed with two or more fluorochromes, obtained bya method comprising: treating polymeric microspheres by adding a polarorganic solvent to an aqueous solution containing the polymericmicrospheres to produce a mixture; and adding at least two fluorochromesto the mixture such that the polymeric microspheres are stained with theat least two fluorochromes, wherein the at least two fluorochromes areselected such that on isolation and excitation of the polymericmicrospheres, a distinct fluorescence signal is emitted from eachfluorochrome, wherein the distinct fluorescence signals have anintensity proportional to the amount of the fluorochrome in thepolymeric microspheres, and wherein the distinct fluorescence signalsare substantially equivalent to targeted fluorescence signals that havebeen used to determine a concentration ratio of the at least twofluorochromes added to the mixture.
 42. The population of claim 41,wherein the polar organic solvent is an aliphatic alcohol, methanol,ethanol, or propanol.
 43. A set of distinct populations of polymericmicrospheres dyed with at least two fluorochromes, wherein the set ofdistinct populations of polymeric microspheres is dyed using a methodcomprising: treating separate populations of polymeric microspheres byadding a polar organic solvent to aqueous solutions containing theseparate populations of polymeric microspheres to produce differentmixtures; preparing a series of solutions, each comprising at least twofluorochromes in a solvent, wherein each of the solutions in the serieshas a different concentration of at least one of the at least twofluorochromes; and adding said each of the solutions to one of thedifferent mixtures to provide the set of distinct populations ofpolymeric microspheres, wherein each of the distinct populations ofpolymeric microspheres emits a different targeted fluorescence intensityfrom the at least two fluorochromes upon excitation, and wherein thedifferent targeted fluorescence intensities have been used to determinethe different concentrations of the at least two fluorochromes includedwithin each of the solutions.
 44. The set of claim 43, wherein the polarorganic solvent is an aliphatic alcohol, methanol, ethanol, or propanol.45. The set of claim 43, wherein the different fluorescence intensity ofsaid each of the distinct populations exhibits a coefficient ofvariation of no greater than about 15 percent.
 46. The set of claim 43,wherein the different fluorescence intensity of said each of thedistinct populations exhibits a coefficient of variation of no greaterthan about 10 percent.
 47. The set of claim 43, wherein the differentfluorescence intensity of said each of the distinct populations exhibitsa coefficient of variation of no greater than about 8 percent.
 48. Theset of claim 43, wherein at least one of the at least two fluorochromesis hydrophobic.
 49. The set of claim 43, wherein the at least twofluorochromes are selected from fluorochromes that emit a red coloredsignal and fluorochromes that emit an orange colored signal.
 50. The setof claim 43, wherein the polymeric microspheres comprise polystyrene,brominated polystyrene, polyacrylic acid, polyacrylonitrile,polyacrylamide, polyacrolein, polydimethylsiloxane, polybutadiene,polyisoprene, polyurethane, polyvinylacetate, polyvinylchloride,polyvinylpyridine, polyvinylbenzylchloride, polyvinyltoluene,polyvinylidene chloride, polydivinylbenzene, polyglycidylmethacrylate,polymethylmethacrylate, or copolymers, blends, composites, orcombinations thereof.
 51. The set of claim 43, wherein the polymericmicrospheres comprise at least one analytical reactant bound covalentlyto functional groups present on a surface of the polymeric microspheresor passively adsorbed to the surface of the polymeric microspheres. 52.The set of claim 43, wherein the polymeric microspheres have a diameterbetween about 10 nm and 100 μm.